Reagents Quantity Reagents Quantity. Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4. Standard 2 Standard Diluent 1 20mL

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1 FOR IN VITRO AND RESEARCH USE ONLY NOT FOR USE IN CLINICAL DIAGNOSTIC PROCEDURES CEA458Ge 96 Tests Enzyme-linked Immunosorbent Assay Kit For Testosterone (Testo) Organism Species: General Instruction manual 11th Edition (Revised in July, 2013) [ INTENDED USE ] The kit is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of testosterone in serum, plasma and other biological fluids. [ REAGENTS AND MATERIALS PROVIDED ] Reagents Quantity Reagents Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard 2 Standard Diluent 1 20mL Detection Reagent A 1 Assay Diluent A 1 12mL Detection Reagent B 1 120μL Assay Diluent B 1 12mL Reagent Diluent 1 300μL Stop Solution 1 6mL TMB Substrate 1 9mL Instruction manual 1 Wash Buffer (30 concentrate) 1 20mL [ MATERIALS REQUIRED BUT NOT SUPPLIED ] 1. Microplate reader with 450 ± 10nm filter. 2. Precision single or multi-channel pipettes and disposable tips. 3. Eppendorf Tubes for diluting samples. 4. Deionized or distilled water. 5. Absorbent paper for blotting the microtiter plate. 6. Container for Wash Solution [ STORAGE OF THE KITS ] 1. For unopened kit: All the reagents should be kept according to the labels on vials. The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 o C upon receipt while the others should be at 4 o C. 2. For opened kit: When the kit is opened, the remaining reagents still need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and reseal along entire edge of zip-seal.

2 Note: It is highly recommended to use the remaining reagents within 1 month provided this is within the expiration date of the kit. For the expiration date of the kit, please refer to the label on the kit box. All components are stable until this expiration date. [ SAMPLE COLLECTION AND STORAGE ] Serum - Allow samples to clot for two hours at room temperature or overnight at 4 o C before centrifugation for 20 minutes at approximately 1000 g. Assay immediately or store samples in aliquot at -20 o C or -80 o C. Avoid repeated freeze/thaw cycles. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 g at 2-8 o C within 30 minutes of collection. Remove plasma and assay immediately or store samples in aliquot at -20 o C or -80 o C for later use. Avoid repeated freeze/thaw cycles. Other biological fluids - Centrifuge samples for 20 minutes at 1000 g. Remove particulates and assay immediately or store samples in aliquot at -20 o C or -80 o C. Avoid repeated freeze/thaw cycles. Note: 1. Samples to be used within 5 days may be stored at 4 o C, otherwise samples must be stored at -20 o C ( 1 month) or -80 o C ( 2 months) to avoid loss of bioactivity and contamination. 2. Sample hemolysis will influence the result, so hemolytic specimen should not be detected. 3. When performing the assay, bring samples to room temperature. [ REAGENT PREPARATION ] 1. Bring all kit components and samples to room temperature (18-25 o C) before use. 2. Standard - Reconstitute the Standard with 0.5mL of Standard Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). The concentration of the standard in the stock solution is 10,000pg/mL. Please prepare 7 tubes containing 0.6mL Standard Diluent and produce a double dilution series according to the picture shown below. Mix each tube thoroughly before the next transfer. Set up 7 points of diluted standard such as 10,000pg/mL, 3,333.3pg/mL, 1,111.1pg/mL, 370.4pg/mL, 123.5pg/mL, and the last EP tubes with Standard Diluent is the blank as 0pg/mL. Tube pg/ml 10,000 3, , Detection Reagent A - Reconstitute the Detection Reagent A with 150μL of Reagent Diluent, kept for 10 minutes at room temperature, shake gently(not to foam). Dilute to the working concentration with Assay Diluent A (1:100). 4. Detection Reagent B - Briefly spin or centrifuge the stock Detection B before use. Dilute to the working concentration with Assay Diluent B (1:100).

3 5. Wash Solution - Dilute 20mL of Wash Solution concentrate (30 ) with 580mL of deionized or distilled water to prepare 600mL of Wash Solution (1 ). 6. TMB substrate - Aspirate the needed dosage of the solution with sterilized tips and do not dump the residual solution into the vial again. Note: 1. Making serial dilution in the wells directly is not permitted. 2. Prepare standard within 15 minutes before assay. Please do not dissolve the reagents at 37 o C directly. 3. Detection Reagent A and B are sticky solutions, therefore, slowly pipette them to reduce the volume errors. 4. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals are completely dissolved. To minimize imprecision caused by pipetting, use small volumes and ensure that pipettors are calibrated. It is recommended to suck more than 10μL for once pipetting. 5. The reconstituted Standards, Detection Reagent A and Detection Reagent B can be used only once. 6. If crystals have formed in the Wash Solution concentrate (30 ), warm to room temperature and mix gently until the crystals are completely dissolved. 7. Contaminated water or container for reagent preparation will influence the detection result. [ SAMPLE PREPARATION ] 1. We are only responsible for the kit itself, but not for the samples consumed during the assay. The user should calculate the possible amount of the samples used in the whole test. Please reserve sufficient samples in advance. 2. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments. Sample should be diluted by 0.01mol/L PBS(PH= ). 3. If the samples are not indicated in the manual, a preliminary experiment to determine the validity of the kit is necessary. 4. Tissue or cell extraction samples prepared by chemical lysis buffer may cause unexpected ELISA results due to the impacts from certain chemicals. 5. Due to the possibility of mismatching between antigen from other origin and antibody used in our kits (e.g., antibody targets conformational epitope rather than linear epitope), some native or recombinant proteins from other manufacturers may not be recognized by our products. 6. Influenced by the factors including cell viability, cell number or sampling time, samples from cell culture supernatant may not be detected by the kit. 7. Fresh samples without long time storage is recommended for the test. Otherwise, protein degradation and denaturalization may occur in those samples and finally lead to wrong results. [ ASSAY PROCEDURE ] 1. Determine wells for diluted standard, blank and sample. Prepare 5 wells for standard points, 1 well for blank. Add 50μL each of dilutions of standard (read Reagent Preparation), blank and samples into the appropriate wells, respectively. And then add 50μL of Detection Reagent A to each well immediately. Shake the plate gently (using a microplate shaker is recommended). Cover with a Plate sealer. Incubate for 1 hour at 37 o C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.

4 2. Aspirate the solution and wash with 350μL of 1X Wash Solution to each well using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher, and let it sit for 1-2 minutes. Remove the remaining liquid from all wells completely by snapping the plate onto absorbent paper. Repeat 3 times. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against absorbent paper. 3. Add 100μL of Detection Reagent B working solution to each well. Incubate for 30 minutes at 37 o C after covering it with the Plate sealer. 4. Repeat the aspiration/wash process for total 5 times as conducted in step Add 90μL of Substrate Solution to each well. Cover with a new Plate sealer. Incubate for minutes at 37 o C (Don't exceed 30 minutes). Protect from light. The liquid will turn blue by the addition of Substrate Solution. 6. Add 50μL of Stop Solution to each well. The liquid will turn yellow by the addition of Stop solution. Mix the liquid by tapping the side of the plate. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 7. Remove any drop of water and fingerprint on the bottom of the plate and confirm there is no bubble on the surface of the liquid. Then, run the microplate reader and conduct measurement at 450nm immediately. Note: 1. Assay preparation: Keep appropriate numbers of wells for each experiment and remove extra wells from microplate. Rest wells should be resealed and stored at -20 o C. 2. Samples or reagents addition:please use the freshly prepared Standard. Please carefully add samples to wells and mix gently to avoid foaming. Do not touch the well wall. For each step in the procedure, total dispensing time for addition of reagents or samples to the assay plate should not exceed 10 minutes. This will ensure equal elapsed time for each pipetting step, without interruption. Duplication of all standards and specimens, although not required, is recommended. To avoid cross-contamination, change pipette tips between additions of standards, samples, and reagents. Also, use separated reservoirs for each reagent. 3. Incubation: To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents are added to the well strips, DO NOT let the strips DRY at any time during the assay. Incubation time and temperature must be controlled. 4. Washing: The wash procedure is critical. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining Wash Solution by aspirating or decanting and remove any drop of water and fingerprint on the bottom of the plate. Insufficient washing will result in poor precision and false elevated absorbance reading. 5. Controlling of reaction time: Observe the change of color after adding TMB Substrate (e.g. observation once every 10 minutes), if the color is too deep, add Stop Solution in advance to avoid excessively strong reaction which will result in inaccurate absorbance reading. 6. TMB Substrate is easily contaminated. Please protect it from light. 7. The environment humidity which is less than 60% might have some effects on the final performance, therefore, a humidifier is recommended to be used at that condition.

5 [ TEST PRINCIPLE ] This assay employs the competitive inhibition enzyme immunoassay technique. A monoclonal antibody specific to testosterone has been pre-coated onto a microplate. A competitive inhibition reaction is launched between biotin labeled testosterone and unlabeled testosterone (Standards or samples) with the pre-coated antibody specific to testosterone. After incubation the unbound conjugate is washed off. Next, avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. The amount of bound HRP conjugate is reverse proportional to the concentration of testosterone in the sample. After addition of the substrate solution, the intensity of color developed is reverse proportional to the concentration of testosterone in the sample. [ CALCULATION OF RESULTS ] This assay employs the competitive inhibition enzyme immunoassay technique, so there is an inverse correlation between testosterone concentration in the sample and the assay signal intensity. Average the duplicate readings for each standard, control, and samples. Create a standard curve on log-log or semi-log graph paper, with the log of testosterone concentration on the y-axis and absorbance on the x-axis. Draw the best fit straight line through the standard points and it can be determined by regression analysis. Using some plot software, for instance, curve expert 1.30, is also recommended. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor. [ TYPICAL DATA ] In order to make the calculation easier, we plot the O.D. value of the standard (X-axis) against the log of concentration of the standard (Y-axis), although concentration is the independent variable and O.D. value is the dependent variable. The O.D. values of the standard curve may vary according to the conditions of assay performance (e.g. operator, pipetting technique, washing technique or temperature effects). Typical standard curve below is provided for reference only. Typical Standard Curve for Testosterone ELISA.

6 [ DETECTION RANGE ] ,000pg/mL. The standard curve concentrations used for the ELISA s were 10,000pg/mL, 3,333.3pg/mL, 1,111.1pg/mL, 370.4pg/mL, 123.5pg/mL. [ SENSITIVITY ] The minimum detectable dose of testosterone is typically less than 51.2pg/mL. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. It was determined by subtracting two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. [ SPECIFICITY ] This assay has high sensitivity and excellent specificity for detection of testosterone. No significant cross-reactivity or interference between testosterone and analogues was observed. Note: Limited by current skills and knowledge, it is impossible for us to complete the cross- reactivity detection between testosterone and all the analogues, therefore, cross reaction may still exist. [ RECOVERY ] Matrices listed below were spiked with certain level of testosterone and the recovery rates were calculated by comparing the measured value to the expected amount of testosterone in samples. Matrix Recovery range (%) Average(%) serum(n=5) EDTA plasma(n=5) heparin plasma(n=5) [ LINEARITY ] The linearity of the kit was assayed by testing samples spiked with appropriate concentration of testosterone and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Sample 1:2 1:4 1:8 1:16 serum(n=5) 80-90% 78-88% 84-98% 87-97% EDTA plasma(n=5) 79-93% % % 81-94% heparin plasma(n=5) 89-99% 88-96% % % [ PRECISION ] Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level testosterone were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level testosterone were tested on 3 different plates, 8 replicates in each plate. CV(%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

7 [ STABILITY ] The stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. To minimize extra influence on the performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is also strongly suggested that the whole assay is performed by the same operator from the beginning to the end. [ ASSAY PROCEDURE SUMMARY ] 1. Prepare all reagents, samples and standards; 2. Add 50μL standard or sample to each well. And then add 50μL prepared Detection Reagent A immediately. Shake and mix. Incubate 1 hour at 37 o C; 3. Aspirate and wash 3 times; 4. Add 100μL prepared Detection Reagent B. Incubate 30 minutes at 37 o C; 5. Aspirate and wash 5 times; 6. Add 90μL Substrate Solution. Incubate minutes at 37 o C; 7. Add 50μL Stop Solution. Read at 450 nm immediately. [ IMPORTANT NOTE ] 1. Limited by the current condition and scientific technology, we can't completely conduct the comprehensive identification and analysis on the raw material provided by suppliers. So there might be some qualitative and technical risks to use the kit. 2. The final experimental results will be closely related to validity of the products, operation skills of the end users and the experimental environments. Please make sure that sufficient samples are available. 3. Kits from different batches may be a little different in detection range, sensitivity and color developing time. Please perform the experiment exactly according to the instruction attached in kit while electronic ones from our website is only for information. 4. Do not mix or substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer. 5. Protect all reagents from strong light during storage and incubation. All the bottle caps of reagents should be covered tightly to prevent the evaporation and contamination of microorganism. 6. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. Do not remove microtiter plate from the storage bag until needed. 7. Wrong operations during the reagents preparation and loading, as well as incorrect parameter setting for the plate reader may lead to incorrect results. A microplate plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 O.D. or greater at 450 ± 10nm wavelength is acceptable for use in absorbance measurement. Please read the instruction carefully and adjust the instrument prior to the experiment. 8. Even the same operator might get different results in two separate experiments. In order to get better reproducible results, the operation of every step in the assay should be controlled. Furthermore, a preliminary experiment before assay for each batch is recommended.

8 9. Each kit has been strictly passed Q.C test. However, results from end users might be inconsistent with our in-house data due to some unexpected transportation conditions or different lab equipments. Intra-assay variance among kits from different batches might arise from above factors, too. 10. Kits from different manufacturers with the same item might produce different results, since we haven t compared our products with other manufacturers. 11. The instruction manual also suits for the kit of 48T, but all reagents of 48T kit are reduced by half. [ PRECAUTION ] The Stop Solution suggested for use with this kit is an acid solution. Wear eye, hand, face, and clothing protection when using this material. [ TROUBLE SHOOTING ] Problem Possible Source Correction Action Poor Standard Curve Poor Precision Low O.D Values Sample Values Improper standard curve preparation Ensure accurate operation of the dilution Incomplete washing and aspiration Adequate washing and adequate aspiration Inaccurate Pipetting Check and Calibrate pipettes Incomplete washing of wells Ensure sufficient washing Inadequate mixing and aspiration reagents Adequate aspiration and mixing reagents Reused pipette tips, containers and sealers Change and use new pipette tips, containers and sealers Inaccurate Pipetting Check and Calibrate pipettes Inadequate reagent volumes added to wells Calibrate pipettes and Add adequate reagents Incorrect incubation times Ensure sufficient incubation times Incorrect incubation temperature Reagents balanced to room temperature Conjugate or substrate reagent failure Mix conjugate &substrate,color should develop immediately No stop solution added Follow the assay protocol in the kit manual Read beyond suggested reading time Read within the time recommended in the manual Improper Sample Storage Store the sample properly and use the fresh sample Improper sample collection and preparation Take proper sample collection and preparation method Low quantity of analyte in samples Use new sample and repeat assay

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